“Do probiotics mitigate GI-induced inflammation and perceived fatigue in athletes? A systematic review”

ABSTRACT Background Fatigue and gastrointestinal (GI) distress are common among athletes with an estimated 30–90% of athletes participating in marathons, triathlons, or similar events experiencing GI complaints. Intense exercise can lead to increased intestinal permeability, potentially allowing members of the gut microbiota to permeate into the bloodstream, resulting in an inflammatory response and cascade of performance-limiting outcomes. Probiotics, through their capacity to regulate the composition of the gut microbiota, may act as an adjunctive therapy by enhancing GI and immune function while mitigating inflammatory responses. This review investigates the effectiveness of probiotic supplementation on fatigue, inflammatory markers, and exercise performance based on randomized controlled trials (RCTs). Methods This review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines and PICOS (Population, Intervention, Comparison, Outcome, Study design) framework. A comprehensive search was conducted in Sportdiscus, PubMed, and Scopus databases, and the screening of titles, abstracts, and full articles was performed based on pre-defined eligibility criteria. Of the 3505 records identified, 1884 were screened using titles and abstracts, of which 450 studies were selected for full-text screening. After final screening, 13 studies met the eligibility criteria and were included for review. The studies contained 513 participants, consisting of 351 males and 115 females, however, two studies failed to mention the sex of the participants. Among the participants, 246 were defined as athletes, while the remaining participants were classified as recreationally active (n = 267). All trials were fully described and employed a double- or triple-blind placebo-controlled intervention using either a single probiotic strain or a multi-strain synbiotic (containing both pro- and pre-biotics). Results This review assesses the effects of daily probiotic supplementation, ranging from 13 to 90 days, on physical performance and physiological markers in various exercise protocols. Ten studies reported improvements in various parameters, such as, enhanced endurance performance, improved anxiety and stress levels, decreased GI symptoms, and reduced upper respiratory tract infections (URTI). Moreover, despite no improvements in maximal oxygen uptake (VO2), several studies demonstrated that probiotic supplementation led to amelioration in lactate, creatine kinase (CK), and ammonia concentrations, suggesting beneficial effects on mitigating exercise-induced muscular stress and damage. Conclusion Probiotic supplementation, specifically at a minimum dosage of 15 billion CFUs daily for a duration of at least 28 days, may contribute to the reduction of perceived or actual fatigue.


Introduction
Endurance sport participation continues to grow globally, with a 49% increase in marathon runners since 2008 [1] and ultra-events growing 57.8% between 1996 and 2018 [2].Endurance exercise involves the prolonged maintenance of constant or self-regulated power over a given distance [3][4][5][6].To sustain exercise performance, athletes require an integration of multiple physiological and psychological systems working in conjunction to regulate exercise intensity and fatigue management [7,8].However, during extended physical exertion, the equilibrium of these systems can be disrupted, detrimentally influencing performance due to factors such as oxidative stress [9], compromised intestinal permeability [10], muscle damage [11], systemic inflammation [12] and immune responses [13].Among these symptoms, gastrointestinal (GI) distress frequently emerges as a consequence, representing a prevalent performance-inhibiting factor with an estimated 30-90% of participants in endurance events experiencing GI complaints [14].However, due to a paucity in the research, the etiology of these symptoms remains elusive [14][15][16].Nonetheless, two physiological theories have been suggested to elucidate the causative factors [17,18].One theory is via the circulatory-gastrointestinal pathway resulting in a redistribution of blood flow to working muscles, reducing oxygen and nutrients to the splanchnic region resulting in splanchnic hypoperfusion, and subsequent ischemia [19][20][21].A second theory is by activation of the neuroendocrine-gastrointestinal pathway, with recent evidence suggesting gut permeability can also influence neural outputs via the "Gut-Brain-Axis" [22].Under certain circumstances, such as during systemic inflammation or infection, pro-inflammatory cytokines can upregulate the enzyme indoleamine 2,3-dioxygenase (IDO), which catalyzes the conversion of tryptophan into Nformyl-kynurenine, initiating the kynurenine pathway resulting in the production of neuroactive compounds such as kynurenic acid and quinolinic acid [23].Kynurenic acid is thought to be a neuroprotective substance, quinolinic acid, conversely, is neurotoxic [24,25].Furthermore, the kynurenine pathway is also involved in the regulation of tryptophan metabolism [25], a key amino acid involved in the synthesis of serotonin, a neurotransmitter that regulates mood and fatigue.Upregulation of quinolinic acid can, therefore, indirectly downregulate serotonin production and thus, affect neural drive contributing to feelings of sadness and increased perceptions of fatigue, potentially influencing physical performance [26][27][28].
Probiotics are live micro-organisms which, when consumed in adequate amounts, confer a health benefit to the host [29] Evidence suggests that probiotics may enhance gut and systemic immune function by improving low-grade inflammation [30,31] and promoting mucosal integrity of the endothelial lining [32].
Probiotics may also aid in maintaining the composition of the microbiota, which encompasses a collective of protozoa, archaea, eukaryotes, viruses, and predominantly bacteria that live symbiotically within humans [33][34][35][36][37]. Several studies have shown that probiotics supplementation could improve immune function in fatigued athletes [38,39] and reduce upper respiratory tract illness (URTI) [40], GI symptoms [38,41] and gut permeability [42].However, it is unclear whether probiotics are effective in mitigating GI-induced inflammation and perceived fatigue in athletes.
This review aims to systematically examine the data from this unique and fast-growing area of research.By assessing and collating RCTs of the highest quality, the findings from multiple studies were analyzed to identify any patterns or relationships between inflammation, probiotic supplementation, and athletic performance.

Search strategy
Studies were identified, screened, and analyzed using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement guidelines [43].Three electronic databases were searched, PubMed, Scopus, and SportDiscus, up to 1 June 2023.The search focused on four main concepts: probiotics, inflammation, fatigue, and exercise.The search incorporated keywords, searched in specific fields (title, abstract, author supplied keywords) and subject headings.A standardized search strategy for key search terms and phrases was combined with Boolean operators to ensure two lists of combination words related to the intervention and outcome of interest, were generated.These included Probiotics/OR Psychobiotics/OR Synbiotics AND Inflammation/OR Inflammatory, Exercise/ OR Athlete AND Fatigue/Or Tiredness [44][45][46][47][48][49].

Screening and data extraction
The titles and abstracts from each database were screened by two authors (R.K and A.McN) to determine eligibility.Following the removal of duplicates, a two-phase search strategy was employed.In the initial phase, the eligibility of the research studies was evaluated in accordance with the PICOS criteria (Appendix A) [50,51].This assessment also involved analysis of subject titles and abstracts, comparing them against an inclusion and exclusion criteria.Studies which had questionable suitability were included with a final decision to keep or remove agreed in phase two.Phase two involved the full articles being retrieved and assessed against an eligibility criterion.Studies were considered eligible if they contained description of participants (athlete or non-athlete), sample size, study design, interventions used (including frequency, dose, strain, and strain designation of probiotic supplementation), and key outcomes of interest (inflammatory biomarkers, performance improvement, and fatigue).Only randomized controlled trials were considered, with the inclusion period spanning from 2012 to 2023 to ensure the inclusion of up-to-date findings and the use of contemporary methods [52,53].The study population consisted of human participants over the age of 18 years.Risk of bias (ROB) was assessed using the latest version of the Cochrane Collaboration risk of bias tool for randomized controlled trials [54].Any differences in opinion relating to study eligibility were resolved through discussion.The study selection process is summarized in Figure 1.

Results
Of the 3505 records identified, 1884 were screened using titles and abstracts, of which 450 studies were refined for full-text screening.After final screening, 13 studies meet the eligibility criteria and were included for review (Figure 1).The studies varied in quality with six RCTs rated as high quality/low rate of bias (ROB) [55][56][57][58][59][60][61] and the remaining seven studies were considered acceptable ROB (Table 1).

Biomarker changes
All 13 studies assessed a range of markers relating to systemic inflammation.CD86 and HLA-DR expression on plasmacytoid dendritic cells (pDCs) are measured as biomarkers of immune activation and maturation, indicating inflammation.Results showed that CD86 expression on pDCs was significantly increased in the LC-Plasma group compared to the placebo [55].However, there were no significant differences in the HLA-DR expression on pDCs [55].Two studies investigated neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR), producing mixed results, with one study [64] showing no significant differences between TWK10 and placebo, whereas the second study [61] investigating TWK10 found a significant decrease in NLR and PLR, indicating reduced inflammation.Multiple studies investigated inflammatory biomarkers including IL-6, IL-8, TNF-α, IL-10, and IgG, with mixed results [56,57,59,60,62,67].L. plantarum PS128 supplementation significantly reduced intense exercise-induced inflammation markers such as TNF-α, IL-6, and IL-8 [62].Another study found no significant changes in IL-6 and CRP values, however, the probiotic group showed lower mean TNF-α values compared to the control group [60].Synbiotic intervention also showed no significant differences in IL-1β and IL-10 concentrations between groups, and immunoglobulin A levels did not show significant variations [67]; moreover, no significant changes were found in sCD14, LR, I-FABP, IL-6, IL-8, IL-10, cortisol, and ACTH [66].
Regarding endotoxin units (EU) and IgG endotoxin-core antibody levels, the probiotic group (LAB4ANTI) exhibited a significant reduction in EU levels in both pre-race and 6 days post-race [59].IgG anti-EU concentrations were significantly lower in the LAB4ANTI group compared to the LAB4 and placebo groups at baseline [59].CK, myoglobin, TBARS, hs-CRP, and testosterone levels were also analyzed as markers of exercise-induced muscle damage and inflammation.The probiotic groups (L-PS23 and HK-PS23) demonstrated significantly lower increases in these markers compared to the placebo group [65].Finally, one study utilized validated psychological scales to evaluate mental well-being, revealing that the probiotic group demonstrated significant reductions in anxiety and stress levels in comparison to the control group, suggesting potential indirect effects on inflammation markers [63].

Fatigue markers
In terms of fatigue assessment, there was great heterogeneity across the studies.All studies included a fatigue element but used different protocols such as subjective questionnaires [55,56,59,63,67], markers of muscle damage or metabolic by-products [55,58,62,65], improvements in performance from baseline [57,59], and RPE [60,61,64,66].Four studies assessed RPE and found that probiotic supplementation improved perceived exhaustion time [60,61,64,66].The studies investigating markers of muscle damage and metabolic by-products such as lactate accumulation, CK, CPK, ammonia, and Tryptophan produced mixed findings [55,56,58,61,62].One study showed CK was significantly reduced following probiotic supplementation, but other indices related to muscular injury (e.g.LDH, protein carbonyl, myoglobin) and fatigue (lactate, ammonia, FFA) remained unchanged after the triathlon competition [62].Whereas another study showed that probiotic supplementation significantly reduced lactate and ammonia concentrations [61].Two studies found no significant difference in CK [56,61].One study found that probiotics reduced TRP degradation rates [58].Two studies assessed fatigue based on performance time compared to baseline scores, with both showing improvements following probiotic supplementation [57,59], however, only one study found performance increased significantly [57].Two studies assessed fatigue symptoms when investigating the occurrence of URTI [55,58], both finding that probiotic supplementation reduced URTI symptoms and, therefore, fatigue by association.Three studies examined GI complaints such as nausea, cramps, diarrhea, stomach pain or discomfort, urge to vomit or defecate, etc., using subjective questionnaires.Two of these studies found a significant improvement [59,66], and the other showed a nonsignificant improvement [60].However, only three of the studies [59,60,66] utilized a form of GI symptom test, one study [66] used a rating scale [68], whereas the other two studies [59,60] based their questionnaires on previously published peer-reviewed journals [69,70].All three studies recorded significantly lower GI symptoms in the groups taking PRO supplementation compared to the control.

Discussion
The aim of this study was to examine the data from high-quality RCTs to identify any patterns or relationships between inflammation, probiotic supplementation, and athletic performance.
Analysis of the 13 studies revealed that some interventions induced positive effects in terms of inflammation, fatigue, and GI symptom reduction.

Effect of probiotics on fatigue
It is well known that fatigue development during endurance performance is largely determined by a complex interplay between psychophysiological and physical capacities [71,72].All studies found a positive correlation between taking probiotic supplementation and a reduction in fatigue, fatigue causing symptoms or perceptions of fatigue.This is in line with existing literature suggesting that probiotics may have positive psychological benefits through interactions with the GBA [73].However, there is great heterogeneity in the methods used to assess fatigue across the studies.For example, several studies assessed fatigue using questionnaires and scales [58][59][60]63,64], such as, Borg's Rate of Perceived Exertion (RPE) [60,64] which is a valid and reliable method for monitoring internal training loads in athletes [74,75].Other scales included "The Brief Fatigue Inventory" (BFI) [67] and "The Perceived Stress Scale (PSS) questionnaire" [63].GI symptom scales were also utilized as they included a subjective fatigue element [59,60,66].Overall, the findings suggest a correlation between decreased GI complaints and reduced perceived fatigue in these studies.
Fatigue was also assessed through biomarker analysis, such as lactate and serum concentrations of TRP and KYN.According to the results, probiotics may offset fatigue by reducing lactate accumulation [64] and TRP degradation [58].Endurance training increases skeletal muscle mitochondria and type 1 fiber content and fatty acid oxidation, which may explain the lower serum lactate due to higher proportion of energy supplied through fatty acid oxidation instead of carbohydrate [76].Reduced TRP degradation supports serotonin metabolism and therefore may reduce perceptions of fatigue [77].Moreover, increased intestinal permeability, psychological stress, reperfusion injury during prolonged exercise, and elevated circulatory proinflammatory cytokines may be a result of higher levels of kynurenines [58].These factors have been associated with negative effects on mood and cognition, which can have implications for athletic performance [78].Additionally, two studies reported a significant reduction in anxiety and stress [63,67].These findings are further reinforced by Adikari and colleagues [79] who observed a notable decrease in competitive anxiety and perceived stress among 20 football players following 8 weeks of daily probiotic supplementation.

Effects of probiotics on inflammation
The studies in this investigation tested a range of inflammatory biomarkers, including inflammatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-1β, TNF-α), ROS, kynurenines, cortisol, biomarkers for gut permeability and muscle damage.Probiotic supplementation produced mixed results, and two studies of various strains produced positive results in reducing inflammatory biomarkers [62,65].However, six studies found no significant changes in pro-inflammatory cytokines following probiotic supplementation [55][56][57][58]60,67].These findings contradict previous studies that have suggested a reduction in pro-inflammatory markers with probiotic intervention [31,80,81].Several factors may contribute to these contrasting results.For instance, the training regimen and exercise protocol employed were not monitored and, therefore, may not have been sufficient to elicit an inflammatory response [55].Moreover, many of the included studies involved athletes who typically exhibit a higher tolerance for high-intensity exercise [55,57,58,60,67] and may not experience the same level of inflammatory response as other participants [51].

Effect of probiotics on performance
The 13 studies examined a range of 'performance' protocols, and VO 2 was not significantly affected through the administration of probiotics, which is indicative of the previous research that attempted to augment VO 2 through nutritional interventions with no success [82,83].Three included studies that recorded an increase in aerobic capacity, which is thought to be a result of regulation of energy balance and metabolism [57,62,63].One of these studies found probiotic supplementation improved endurance performance significantly by 130% which the authors suggest may be a result of probiotics ameliorating the onset of central and peripheral fatigue mechanisms.The influence of probiotics on aerobic capacity is consistent with a previous review, which reported an increase in oxygen uptake among swimmers who consumed probiotics [84].These findings contribute to the existing body of research that demonstrates a positive association between probiotic supplementation and a decrease in time to fatigue.This correlation has been observed in preclinical studies [85][86][87] as well as clinical studies [64,88,89] involving both athletes and non-athletes.

GI complaints
GI complaints are common in endurance sports [9,90] with various degrees of severity, from mild reflux and nausea to vomiting and bloody diarrhea [16,91].However, only three of the studies [59,60,66] utilized a form of GI symptom test, with Pugh and colleagues [66] using a rating scale [68], whereas the other two studies [59,60] based their questionnaires on previously published peer-reviewed articles [69,70].All three studies recorded lower GI symptoms in the groups taking probiotic supplementation compared to the control, two significantly [59,66] and one non-significantly [60].These three studies all used multi-strain probiotics, with CFUs ranging from 15 to 30 billion per dosage for durations between 28 and 90 days.A previous review [38] investigating the efficacy of probiotic supplementation in reducing GI symptoms in athletes produced similar findings, reporting frequency, and severity of GI symptoms were reduced by approximately one-third in athletes supplementing with a multi-strain Lactobacillus or Bifidobacterium probiotic.

Limitations
Several limitations should be considered when interpreting these findings.Heterogeneity in exercise protocols and daily training regimens across the studies introduces variability in participants' training status, potentially reflecting different stages in their training cycles.Certain studies also lacked specific assessments, for example, some studies did not measure biomarkers of gut permeability or specific markers of muscle damage [55,56,62,64,65,67].These assessments could provide valuable insights into the mechanisms underlying the influence of probiotics.Diet standardization was another limitation.While a few studies set limitations on additional supplements, alternative probiotics, fermented foods, and antibiotics, overall diet standardization was lacking [57,64,66,67].Moreover, dietary intake during races was not recorded, one study [66] did not monitor the use of carbohydrate and water intake, which has been shown in the previous research to have influenced performance outcome due to variability with gastric emptying [92].The assessment of fatigue also exhibited heterogeneity across the studies.Different protocols and measures were used, making it challenging to compare and generalize the findings related to fatigue.
The search strategy employed in this systematic review focused solely on three electronic databases (PubMed, Scopus, and SportDiscus), potentially excluding relevant studies from other sources and introducing a selection bias.However, the databases selected are robust and contained all peer reviewed high-quality papers.Moreover, the use of PRISMA guidelines ensured a systematic approach to identification and analysis.
Although the study population consisted of a total of 513 participants, there was an imbalance in the distribution between male (351) and female (115) participants.Additionally, two studies did not report the gender of the participants, which limits the generalizability of the findings between males and females.However, the inclusion of a diverse participant population, including both athletes and recreationally active individuals, broadens the applicability of the study findings within these specific populations.
Finally, the probiotic strain specificity and dose dependency of probiotics' effects were not fully elucidated in our analysis.A nuanced classification of probiotics, based on their established or proposed impacts on inflammation, oxidative stress, or gut health, might elucidate their prospective advantages in enhancing exercise and athletic performance.Additionally, one study [60] indicated at least (≥) 4.3 × 10^9 CFU for various strains, introducing ambiguity in the precise quantity of CFUs provided.This range represents a limitation in the clarity and reproducibility of the probiotic dosages administered, potentially influencing the reviews outcomes and interpretations.

Conclusion
This comprehensive review highlights the potential beneficial effects of probiotic supplementation on blood biomarkers, physical performance, and fatigue.The findings suggest that probiotics, specifically a multi-strained probiotic at a minimum dosage of 15 billion CFUs daily for a duration of at least 28 days, may contribute to the reduction of perceived or actual fatigue.These findings also align with existing literature suggesting that probiotics may exert psychological benefits through their interactions with the gut-brain axis.Notwithstanding, it is important to acknowledge the limitations present in the selected studies, including disparities in probiotic strains, timing, dosage, duration, and testing protocols, as well as the lack of standardized training regimes.Future research should aim to address these limitations, establish standardized protocols, and explore the mechanisms underlying probiotic effects to optimisetheir utilisation for enhancing exercise performance.

Disclosure statement
No potential conflict of interest was reported by the authors.

Identification of studies via databases and registers Identification Screening Included Figure 1. Preferred
reporting items for systematic reviews and meta-analysis study flow diagram.